Research in the field of molecular biology has revealed that the genetic origin and functional activity of a cell can be deduced from the study of its ribonucleic acid (RNA). This information may be of use in clinical practice, to diagnose infections, to detect the presence of cells expressing oncogenes, to detect familial disorders, to monitor the state of host defense mechanisms and to determine the HLA type or other marker of identity.
Current methods for isolating RNA include a variety of techniques to disrupt the cell and liberate RNA into solution and to protect RNA from RNases.
Thereafter, the RNA is separated from the DNA and protein which is solubilized along with the RNA. The use of the powerfully chaotropic salts of guanidinium to simultaneously lyse cells, solubilize RNA and inhibit RNases was described in Chirgwin et al, Biochem., 18:5294-5299 (1979). Other methods free solubilized RNA of contaminating protein and DNA by extraction with phenol at an acidic pH using chloroform to effect a phase separation [D. M. Wallace, Meth. Enzym., 15:33-41 (1987)]. A commonly used single step isolation of RNA involves homogenizing cells in 4M guanidinium isothiocyanate, followed by the sequential addition of sodium acetate (pH 4) phenol, and chloroform/isoamyl alcohol. After centrifugation, RNA is precipitated from the upper layer by the addition of alcohol [P. Chomczynski and N. Sacchi, Anal. Biochem., 162:156-159 (1987) and "Preparation and Analysis of RNA" in Current Protocols in Molecular Biology, Unit 4.2 (Supplement 14), ed. F. M. Ausubel et al, John Wiley, (1991)]. Less commonly used methods include the addition of hot phenol to a cell suspension, followed by alcohol precipitation [T. Maniatis et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982)]; the use of anionic or non-ionic surfactants to lyse cells and liberate cytoplasmic RNA; and the use of inhibitors of RNases such as vanadyl riboside complexes and diethylpyrocarbonate [L. G. Davis et al, "Guanidine Isothiocyanate Preparation of Total RNA" and "RNA Preparation: Mini Method" in Basic Methods in Molecular Biology, Elsevier, New York, pp. 130-138 (1991). U.S. Pat. No. 4,843,155, Chomczynski, describes a method in which a stable mixture of phenol and guanidinium salt at an acidic pH is added to the cells. After phase separation with chloroform, the RNA in the aqueous phase is recovered by precipitation with an alcohol.
The ability of cationic surfactants to lyse cells and simultaneously precipitate RNA and DNA from solution was described in U.S. Pat. No. 5,010,183, by Macfarlane. The '183 patent's method differs fundamentally from those described above in that its first step renders the RNA insoluble, whereas in the above described methods the first step is to solubilize RNA. In the preferred method of the '183 patent, a 2% solution of the surfactant benzyldimethyl-n-hexadecylammonium chloride together with 40% urea and other additives is added to a cell suspension, and the mixture is centrifuged. The pellet is resuspended in ethanol, from which the RNA and DNA is precipitated by the addition of a salt. In attempts to apply this method to blood, the inventor found that the use of the latter surfactant and other commercially available surfactants results in inefficient precipitation of RNA and incomplete lysis of blood cells [see Tables I and II, below]. There is a need for improved cationic surfactants for this purpose.
Current methods for analyzing RNA in blood use amplification methods (including the polymerase chain reaction), and are capable of detecting the presence of specific sequences of RNA present in minute amounts. Investigators wishing to study RNA in white blood cells are likely to separate these cells from blood by centrifugal methods (typically through a gradient of Ficoll/hypaque), and then apply one of the above described methods to the isolated cells. Thus, there is no established method for isolating RNA from whole blood. Similarly, investigators wishing to study viruses may separate viral RNA from plasma using such methods.
Even in view of these known methods, the use of RNA in clinical practice is hampered by the difficulty of separating RNA from the protein and DNA in the cell before the RNA is degraded by nucleases, such as RNase. RNase and other nucleases are present in blood in sufficient quantities to destroy unprotected RNA in a few seconds. Successful methods for the isolation of RNA from cells must be capable of preventing hydrolysis of RNA by nucleases.
There remains a need in the art for a simple method for isolating RNA from blood, other fluids and cells, which method minimizes hydrolysis and degradation of the RNA so that isolated RNA can be used in clinical studies.